Angiogenesis is a critical process in the formation of microvasculature to deliver nutrients and oxygen to target cells and tissues. During this process, endothelial cells respond to specific extracellular signals that cause them to migrate from existing vessels and form tubules through a process called tubulogenesis. Alterations or disruptions in these signaling mechanisms, though, can lead to the formation of unhealthy vessel structures, indicative of disease states (i.e. cancers). In this proposal, we aim to use micropatterned biomaterials to control the spatiotemporal elements of endothelial cell microenvironments, composed primarily of adhesive, mechanical, and diffusible/soluble cues. By observing and characterizing how endothelial cells manipulate and coordinate responses from their local microenvironment, we can classify their corresponding cellular phenotypes and tubule networks based upon their specific interactions with individual cues. To accomplish this, we will first create a two-photon-based patterning strategy capable of immobilizing multiple biomolecules in parallel within three dimensional (3D) poly(ethylene glycol) (PEG) hydrogels through the use of orthogonal photochemistries. In addition, we will incorporate new functionalities to allow the bulk hydrogel properties to be transiently modified. Using this technology we will create patterns of adhesive ligands within the hydrogel with bulk and localized (patterned) growth factors. By first controlling the spatial introduction of growth factos, we will investigate how we can control the initiation of tubulogenic events (i.e. branching) in 3D. Furthermore, by employing the growth factors involved in wound healing: platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF), we will investigate how the order in which each growth factor is encountered as well as the display (i.e., bulk or locally immobilized) of the individual growth factors effects the relatie structure of the tubule network. Finally, we will explore the temporal introduction of these growth factors and how their incorporation during tubulogenesis can alter, disrupt, or reinforce endothelial cell responses. From these studies, we anticipate that we can control the branching, elongation, and overall structure of the tubules that are formed. To verify this we will create a comprehensive method to characterize and classify tubule networks as well as the phenotype of the endothelial cells, themselves. Finally, the variations in the spatial and temporal introduction should enable us to decouple these effects to create a tubulogenic model which will allow us to pre-program 3D cellular microenvironments to drive specific tubulogenic and phenotypic outcomes.